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HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells.

Chavez L, Calvanese V, Verdin E - PLoS Pathog. (2015)

Bottom Line: Importantly, returning productively infected cells to a resting state is not associated with a significant silencing of the integrated HIV.We further show that resting CD4+ T cells from human lymphoid tissue (tonsil, spleen) show increased latency after infection when compared to peripheral blood.Our findings raise significant questions regarding the most commonly accepted model for the establishment of latent HIV and suggest that infection of both resting and activated primary CD4+ T cells produce latency.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Virology and Immunology, San Francisco, California, United States of America; Biomedical Sciences Program, University of California, San Francisco, San Francisco, California, United States of America.

ABSTRACT
Highly active antiretroviral therapy (HAART) suppresses human immunodeficiency virus (HIV) replication to undetectable levels but cannot fully eradicate the virus because a small reservoir of CD4+ T cells remains latently infected. Since HIV efficiently infects only activated CD4+ T cells and since latent HIV primarily resides in resting CD4+ T cells, it is generally assumed that latency is established when a productively infected cell recycles to a resting state, trapping the virus in a latent state. In this study, we use a dual reporter virus--HIV Duo-Fluo I, which identifies latently infected cells immediately after infection--to investigate how T cell activation affects the establishment of HIV latency. We show that HIV latency can arise from the direct infection of both resting and activated CD4+ T cells. Importantly, returning productively infected cells to a resting state is not associated with a significant silencing of the integrated HIV. We further show that resting CD4+ T cells from human lymphoid tissue (tonsil, spleen) show increased latency after infection when compared to peripheral blood. Our findings raise significant questions regarding the most commonly accepted model for the establishment of latent HIV and suggest that infection of both resting and activated primary CD4+ T cells produce latency.

No MeSH data available.


Related in: MedlinePlus

Primary CD4+ T cells transitioning from an activated state back to a resting state are more likely to become latently infected.(A) Schematic of experimental procedure. Primary CD4+ T cells were isolated from uninfected donor blood and stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 72 h and were then allowed to return to a resting state over 20 days in the presence of IL-2. Cells were infected at peak activation (day 4) and every 5 days thereafter as they returned to a resting state. (B) Expression of activation markers CD69 and CD25 as the cells transition from an activated state to a resting state. Flow cytometry was performed 72 h post activation and every 5 days after the activation beads were removed. Data shown are from a single donor, but representative of three separate donors. Percentage of live cells is calculated from the live gate (forward vs side scatter plots) in the FACS analysis and represents the average of three donors. (C) Quantified values of the cells’ activation status from panel B. Data represents the average of three donors. (D) Infection profiles of primary CD4+ T cells as they transition back to a resting state. Cells were spinoculated with HIV Duo-Fluo I 72 h after activation and every 5 days after the activation beads were removed. Infection was analyzed by flow cytometry 72 h post-infection. Data shown are from a single donor, but representative of three separate donors. (E) Quantified values of latent infection and productive infection from panel D. Data represents the average of three donors. (F) Ratios of latent infection to productive infection were calculated using data from panel E. Data represents the average of three donors. *, P < 0.05; **, P < 0.01; ns, non-significant.
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ppat.1004955.g002: Primary CD4+ T cells transitioning from an activated state back to a resting state are more likely to become latently infected.(A) Schematic of experimental procedure. Primary CD4+ T cells were isolated from uninfected donor blood and stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 72 h and were then allowed to return to a resting state over 20 days in the presence of IL-2. Cells were infected at peak activation (day 4) and every 5 days thereafter as they returned to a resting state. (B) Expression of activation markers CD69 and CD25 as the cells transition from an activated state to a resting state. Flow cytometry was performed 72 h post activation and every 5 days after the activation beads were removed. Data shown are from a single donor, but representative of three separate donors. Percentage of live cells is calculated from the live gate (forward vs side scatter plots) in the FACS analysis and represents the average of three donors. (C) Quantified values of the cells’ activation status from panel B. Data represents the average of three donors. (D) Infection profiles of primary CD4+ T cells as they transition back to a resting state. Cells were spinoculated with HIV Duo-Fluo I 72 h after activation and every 5 days after the activation beads were removed. Infection was analyzed by flow cytometry 72 h post-infection. Data shown are from a single donor, but representative of three separate donors. (E) Quantified values of latent infection and productive infection from panel D. Data represents the average of three donors. (F) Ratios of latent infection to productive infection were calculated using data from panel E. Data represents the average of three donors. *, P < 0.05; **, P < 0.01; ns, non-significant.

Mentions: Because HIV replicates most efficiently in activated CD4+ T cells [23], and the largest in vivo latent reservoir is within resting memory CD4+ T cells, we next investigated whether HIV latency is preferentially established in CD4+ T cells that become infected as they transition from an activated to a resting state. To do this, we isolated total CD4+ T cells from peripheral blood of uninfected donors and stimulated the cells with αCD3/αCD28 activating beads in the presence of IL-2 for 3 days (Fig 2A). We then removed the αCD3/αCD28 activating beads and allowed the cells to return to a resting state in the presence of IL-2 for 20 days. We infected the CD4+ T cells with HIV Duo-Fluo I at peak activation (day 4) and every 5 days thereafter as they transitioned back to resting. As indicated by expression of the activation markers CD69 and CD25, the cells transitioned from active to resting over the 20 days and remained >60% viable (Fig 2B). Maximal activation occurred at day 4 with 79% of cells expressing both CD69 and CD25 (Fig 2C). By day 9, however, 31% of cells no longer expressed CD69 or CD25, and by day 24, 92% of cells no longer expressed either activation marker. Despite most cells losing CD69 expression by day 14 (<1% CD69+), a small fraction of cells continued to express CD25 throughout the experiment—with 8% of cells still CD25+ at day 24—suggesting that while most CD4+ T cells had returned to a resting state by day 24, a small population was still transitioning back to a resting state. Others have observed similar expression profiles while trying to return activated CD4+ T cells to a resting state [52, 53].


HIV Latency Is Established Directly and Early in Both Resting and Activated Primary CD4 T Cells.

Chavez L, Calvanese V, Verdin E - PLoS Pathog. (2015)

Primary CD4+ T cells transitioning from an activated state back to a resting state are more likely to become latently infected.(A) Schematic of experimental procedure. Primary CD4+ T cells were isolated from uninfected donor blood and stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 72 h and were then allowed to return to a resting state over 20 days in the presence of IL-2. Cells were infected at peak activation (day 4) and every 5 days thereafter as they returned to a resting state. (B) Expression of activation markers CD69 and CD25 as the cells transition from an activated state to a resting state. Flow cytometry was performed 72 h post activation and every 5 days after the activation beads were removed. Data shown are from a single donor, but representative of three separate donors. Percentage of live cells is calculated from the live gate (forward vs side scatter plots) in the FACS analysis and represents the average of three donors. (C) Quantified values of the cells’ activation status from panel B. Data represents the average of three donors. (D) Infection profiles of primary CD4+ T cells as they transition back to a resting state. Cells were spinoculated with HIV Duo-Fluo I 72 h after activation and every 5 days after the activation beads were removed. Infection was analyzed by flow cytometry 72 h post-infection. Data shown are from a single donor, but representative of three separate donors. (E) Quantified values of latent infection and productive infection from panel D. Data represents the average of three donors. (F) Ratios of latent infection to productive infection were calculated using data from panel E. Data represents the average of three donors. *, P < 0.05; **, P < 0.01; ns, non-significant.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4466167&req=5

ppat.1004955.g002: Primary CD4+ T cells transitioning from an activated state back to a resting state are more likely to become latently infected.(A) Schematic of experimental procedure. Primary CD4+ T cells were isolated from uninfected donor blood and stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 72 h and were then allowed to return to a resting state over 20 days in the presence of IL-2. Cells were infected at peak activation (day 4) and every 5 days thereafter as they returned to a resting state. (B) Expression of activation markers CD69 and CD25 as the cells transition from an activated state to a resting state. Flow cytometry was performed 72 h post activation and every 5 days after the activation beads were removed. Data shown are from a single donor, but representative of three separate donors. Percentage of live cells is calculated from the live gate (forward vs side scatter plots) in the FACS analysis and represents the average of three donors. (C) Quantified values of the cells’ activation status from panel B. Data represents the average of three donors. (D) Infection profiles of primary CD4+ T cells as they transition back to a resting state. Cells were spinoculated with HIV Duo-Fluo I 72 h after activation and every 5 days after the activation beads were removed. Infection was analyzed by flow cytometry 72 h post-infection. Data shown are from a single donor, but representative of three separate donors. (E) Quantified values of latent infection and productive infection from panel D. Data represents the average of three donors. (F) Ratios of latent infection to productive infection were calculated using data from panel E. Data represents the average of three donors. *, P < 0.05; **, P < 0.01; ns, non-significant.
Mentions: Because HIV replicates most efficiently in activated CD4+ T cells [23], and the largest in vivo latent reservoir is within resting memory CD4+ T cells, we next investigated whether HIV latency is preferentially established in CD4+ T cells that become infected as they transition from an activated to a resting state. To do this, we isolated total CD4+ T cells from peripheral blood of uninfected donors and stimulated the cells with αCD3/αCD28 activating beads in the presence of IL-2 for 3 days (Fig 2A). We then removed the αCD3/αCD28 activating beads and allowed the cells to return to a resting state in the presence of IL-2 for 20 days. We infected the CD4+ T cells with HIV Duo-Fluo I at peak activation (day 4) and every 5 days thereafter as they transitioned back to resting. As indicated by expression of the activation markers CD69 and CD25, the cells transitioned from active to resting over the 20 days and remained >60% viable (Fig 2B). Maximal activation occurred at day 4 with 79% of cells expressing both CD69 and CD25 (Fig 2C). By day 9, however, 31% of cells no longer expressed CD69 or CD25, and by day 24, 92% of cells no longer expressed either activation marker. Despite most cells losing CD69 expression by day 14 (<1% CD69+), a small fraction of cells continued to express CD25 throughout the experiment—with 8% of cells still CD25+ at day 24—suggesting that while most CD4+ T cells had returned to a resting state by day 24, a small population was still transitioning back to a resting state. Others have observed similar expression profiles while trying to return activated CD4+ T cells to a resting state [52, 53].

Bottom Line: Importantly, returning productively infected cells to a resting state is not associated with a significant silencing of the integrated HIV.We further show that resting CD4+ T cells from human lymphoid tissue (tonsil, spleen) show increased latency after infection when compared to peripheral blood.Our findings raise significant questions regarding the most commonly accepted model for the establishment of latent HIV and suggest that infection of both resting and activated primary CD4+ T cells produce latency.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Virology and Immunology, San Francisco, California, United States of America; Biomedical Sciences Program, University of California, San Francisco, San Francisco, California, United States of America.

ABSTRACT
Highly active antiretroviral therapy (HAART) suppresses human immunodeficiency virus (HIV) replication to undetectable levels but cannot fully eradicate the virus because a small reservoir of CD4+ T cells remains latently infected. Since HIV efficiently infects only activated CD4+ T cells and since latent HIV primarily resides in resting CD4+ T cells, it is generally assumed that latency is established when a productively infected cell recycles to a resting state, trapping the virus in a latent state. In this study, we use a dual reporter virus--HIV Duo-Fluo I, which identifies latently infected cells immediately after infection--to investigate how T cell activation affects the establishment of HIV latency. We show that HIV latency can arise from the direct infection of both resting and activated CD4+ T cells. Importantly, returning productively infected cells to a resting state is not associated with a significant silencing of the integrated HIV. We further show that resting CD4+ T cells from human lymphoid tissue (tonsil, spleen) show increased latency after infection when compared to peripheral blood. Our findings raise significant questions regarding the most commonly accepted model for the establishment of latent HIV and suggest that infection of both resting and activated primary CD4+ T cells produce latency.

No MeSH data available.


Related in: MedlinePlus